Use of prosaposin and neurotrophic peptides derived therefrom

ABSTRACT

Prosaposin, saposin C and various peptide fragments of saposin C stimulate neurite outgrowth in vitro. In addition, prosaposin and saposin C promote increased myelination ex vivo. Prosaposin is present in large neurons of the brain, including both upper and lower motor neurons.

GOVERNMENT SUPPORT

This research was supported by National Institutes of Health Grant NS08682. The government may have certain rights in this invention.

This application is a divisional of U.S. patent application Ser. No.08/100,247, filed Jul. 30, 1993, by the same inventor, now U.S. Pat. No.5,571,787.

FIELD OF THE INVENTION

This invention discloses the utilization of prosaposin, or fragmentsthereof, as a cytokine, growth factor or neurotrophin. Morespecifically, prosaposin may be a neurotrophic factor for populations ofneurons for which neurotrophins have yet to be identified.

BACKGROUND OF THE INVENTION

Prosaposin, a 70 kilodalton glycoprotein, is the precursor of a group offour small heat-stable glycoproteins which are required for hydrolysisof glycosphingolipids by lysosomal hydrolases (Kishimoto et al., (1992)J. Lipid Res., 33: 1255-1267) Prosaposin is proteolytically processed inlysosomes to generate saposins A, B, C, and D which exist as fouradjacent tandem domains in prosaposin (O'Brien and Kishimoto, (1991)FASEB J., 5: 301-308) All four saposins are structurally similar to eachother, including the placement of six cysteines, a glycosylation siteand conserved proline residues.

Unprocessed prosaposin also exists as an integral membrane protein and asecreted protein which is present in human milk, cerebrospinal fluid andseminal plasma. The presence of high concentrations of unprocessedprosaposin in the central nervous system indicates that it may play asignificant role in addition to activation of lysosomal hydrolases.

Prosaposin binds membrane lipids called glycosphingolipids which aresphingolipids consisting of a carbohydrate head group and twohydrocarbon chains; a fatty acid and a sphingosine derivative.Glycosphingolipids are important components of the myelin sheath, astructure which protects and insulates nerve fibers. Demyelination is adefect common to a number of central nervous system disorders, the mostcommon being multiple sclerosis (MS). MS, a chronic disorder which maylead to total disability, is characterized by damage to the myelinsheath leaving the axons mostly intact. It is currently believed thatautoimmune mechanisms, perhaps vitally-induced, may play a role indevelopment of the disease. There is currently no effective treatmentfor MS. Other central nervous system disorders involving demyelinationinclude acute disseminated encephalomyelitis, amyotrophic lateralsclerosis, acute necrotizing hemorrhagic leukodystrophy, progressivemultifocal leukoencephalitis, metachromatic leukodystrophy and adrenalleukodystrophy. An example of a demyelinating disease of the peripheralnervous system is Guillain-Barre syndrome (Pathologic Basis of Disease,Robbins, S. L. and Cotran, R. S., eds, W. B. Saunders, Philadelphia,(1979), pp. 1578-1582).

Post-polio syndrome is characterized by muscle fatigue and decreasedendurance with accompanying muscle weakness and atrophy. The disease isbelieved to be caused in part by the same type of spinal cord motorneurons that are damaged in amyotrophic lateral sclerosis.

Peripheral nerve injuries and peripheral neuropathies, such as thoseresulting from diabetes or chemotherapy, comprise the most prevalentperipheral nervous system disorders (see Table 1) Current treatments forperipheral nerve disorders only treat the symptoms, not the cause of thedisease.

                  TABLE 1                                                         ______________________________________                                        Disease          No. of U.S. patients                                         ______________________________________                                        Amyotrophic Lateral                                                                             30,000                                                      Sclerosis                                                                     Spinal Muscular Atrophy                                                                         50,000                                                      Post-Polio Syndrome                                                                            250,000                                                      Guillain-Barre Syndrome                                                                         20,000                                                      Muscular Dystrophies                                                                           175,000                                                      Peripheral Neuropathies                                                                        1,000,000                                                    Peripheral Nerve Injuries                                                                      500,000                                                      Total            2,150,000                                                    ______________________________________                                    

Prosaposin binds glycosphingolipids such as gangliosides, cerebrosidesand sulfatides with high affinity and facilitates their transfer frommicelles to membranes (Sueda, et al. (1993) J. Biol. Chem. in press;Hiraiwa et al., (1992) Proc. Natl. Acad. Sci. USA., 89: 11254-11258).Gangliosides contain one or more sialic acid residues and are mostabundant in the plasma membrane of neurons where they constituteapproximately 6% of the total lipid mass. Although the function ofgangliosides is largely unknown, they have been implicated in thestimulation of neuronal differentiation, neuritogenesis and nervoussystem repair.

Neurotrophins may be defined as those proteins capable of affecting thesurvival, target innervation and/or function of neuronal cellpopulations (Barde, (1989) Neuron, 2: 1525-1534). The efficacy ofneurotrophins both in vitro and in vivo has been well-documented. Themost well-characterized of such proteins is nerve growth factor (NGF)which is synthesized by target cells of sympathetic and sensory neuronsand acts as a trophic factor for forebrain cholinergic, peripheral andsensory neurons (Hefti et al., (1989) Neurobiol. Aging, 10: 515-533). Invivo experiments indicate that NGF can reverse naturally-occurring aswell as physical traumatic injuries to peripheral nerves. For example,local application of NGF has been shown to prevent the atrophy ofsensory ganglia resulting from transection of the sciatic nerve in adultrats (Rich et al., (1987) J. Neurocytol., 16: 261-268). In addition, NGFplays a role in the neural regenerative process since it enhancesneurite extension of developing sympathetic and sensory neurons (Putyeset al., (1988) Nature, 336: 123-128). Moreover, since NGF supports thefunction of forebrain cholinergic neurons which are lost in Alzheimer'spatients, this indicates that NGF may have a clinical use in treatmentof this disease (Hefti et al., (1989) Neurobiol. Aging, 10: 515-533).

Brain-Derived Neurotrophic Factor (BDNF) is synthesized in the centralnervous system and is a trophic factor for peripheral sensory neurons,dopaminergic neurons of the substantia nigra, central cholinergicneurons and retinal ganglia (Henderson et al., (1993) Restor. Neurol.Neurosci., 5: 15-28). BDNF has also been shown to preventnormally-occurring cell death both in vitro and in vivo (Hofer andBarde, (1988) Nature, 331: 261-262).

Since NGF and BDNF share large regions of homology (approximately 50%),degenerate oligonucleotide primers corresponding to four of theseregions were used in PCR reactions to amplify novel related sequences. Arelated neurotrophic factor called neurotrophin 3 (NT-3) was cloned(Maisonpierre et al., (1990) Science, 247: 1446-1451). NT-3 is foundboth centrally and peripherally and is capable of promoting survival ofsensory and sympathetic neurons, including dorsal root ganglia (DRG)explants.

The three neurotrophins described above have different neuronalspecificities. All three neurotrophins induced neurite outgrowth fromDRG explants. NGF induces neurite outgrowth from sympathetic ganglia(SG) but not nodose ganglion (NG), whereas BDNF induces neuriteoutgrowth from NG but not SG. NT-3 promotes neurite outgrowth from NGand to a lesser extent from SG, suggesting a broader specificity thaneither NGF or BDNF (Lindsay et al., (1991) Restor. Neurol. Neurosci., 2:211-220).

Ciliary Neurotrophic Factor (CNTF) promotes survival of chicken embryociliary ganglia in vitro and was also found to support survival ofcultured sympathetic, sensory and spinal motor neurons (Ip et al.,(1991) J. Physiol., Paris, 85: 123-130). Local administration of thisprotein to the lesion site of newborn rats has been shown to prevent thedegeneration of the corresponding motor neurons. CNTF also rescued motorneurons from developmental cell death (Henderson et al., (1993) Restor.Neurol. Neurosci., 5: 15-28).

Fibroblast Growth Factor (FGF) can also promote in vitro survival ofembryonic neurons. Effects have also been observed on neuronal survivalafter lesion in vivo. FGF apparently acts on a wide variety of neurons(Hefti et al., (1989) Neurobiol. Aging, 10: 515-533).

The identification of prosaposin itself as a neurotrophic factor whichis present in the cell bodies of large populations of neurons includingupper and lower motor neurons, and its ability to induce myelination inmouse cerebellar explants, represent significant new functions for thisprotein. Additionally, the fact that fragments of prosaposin retainneuritogenic activity has no precedent in the literature. No reportshave appeared on the use of small active fragments of the aforementionedneurotrophic factors to promote neuronal survival and differentiation.Prosaposin and its derivatives are therefore believed to have importanttherapeutic potential in the treatment of neurodegenerative anddemyelination disorders.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1a is a graph illustrating the neurite outgrowth response of NS20Yneuroblastoma cells treated with prosaposin, saposin C, active 22-merpeptide (CEFLVKEVTKLIDNNKTEKEIL; SEQ ID NO: 1) and iodine labeled 18 metover the 0.01-0.5 μg/ml range. The concentration of effector protein, inμg/ml, is shown on the x-axis and the percentage of cells with neuritesis shown on the y-axis.

FIG. 1b is a bar graph showing the effect of 5 μg/ml NGF on neuriteoutgrowth in prosaposin and saposin C treated NS20Y cells. The y-axisindicates the percentage of cells with neurites.

FIG. 2a shows a hydropathy plot of the human saposin C sequence. Theamino acid residue position is shown on the x-axis and the hydropathicindex is shown on the y-axis.

FIG. 2b provides a sequence alignment of the active 22-mer human saposinC sequence (SEQ ID NO: 1) with the same sequence from four otherspecies: mouse (SEQ ID NO: 6), rat (SEQ ID NO: 7), guinea pig (SEQ IDNO: 8), and bovine (SEQ ID NO: 9). The consensus (completely conserved)residues are indicated below the sequence alignment as SEQ ID NO: 10.The sequence of human saposin A (SEQ ID NO: 11) (which is inactive) inthe same region is provided to illustrate the divergence between thesequence of three of the first four residues in the same hydrophilicregion (18-29) in saposin A but conservation of the remaining residues.

SUMMARY OF THE INVENTION

One embodiment of the present invention is a method for stimulatingneural cell outgrowth or increased myelination by contacting neuronalcells with a composition including prosaposin or a fragment thereofhaving the ability to promote increased neural outgrowth or increasedmyelination activity. Preferably the prosaposin is native. Mostpreferably the prosaposin is recombinantly produced. The neurotrophicfragment may advantageously be saposin C, a peptide comprising aminoacids 8-29 of saposin C, or another peptide containing the same activeregion. Most preferably, this fragment consists essentially of theactive neurotrophic fragment located within amino acids 8-29 of SEQ IDNO 1. Preferably the neuronal cells are either NS20Y, Neuro 2A or N1E115neuroblastoma cells. These neuronal cells are preferably contacted invitro and most preferably contacted in vivo. Also within the scope ofthe invention are cells from mouse cerebellar explants.

Another aspect of the present invention relates to a method fortreatment of demyelination disorders in a mammal by identifying a mammalafflicted with the disorder, and administering to the mammal apharmaceutically effective demyelination inhibiting amount of prosaposinor a neurotrophic fragment thereof. Preferably this fragment is saposinC and the demyelination disorder is either multiple sclerosis, acutedisseminated leukoencephalitis, progressive multifocal leukoencephalitisor adrenal leukodystrophy. Preferably, the method of administration iseither intramuscular, intradermal, subcutaneous, intracranial,intracerebrospinal or topical in a biologically compatible carrier. Mostpreferably, the administration is intravenous. Moreover, the prosaposinor fragment thereof may be advantageously enclosed in a lamellarstructure.

The invention further comprises a method for halting or slowing theprogress of neural or myelin degeneration in neural tissue, bycontacting neuronal tissue susceptible to such degradation withprosaposin or an active degradation-inhibiting fragment thereof.Preferably, the fragment is saposin C and the tissue is in vivo. Mostpreferably the fragment is the neurotrophic peptide and the tissue is invivo.

Another aspect of the present invention is a method for the treatment ofneuronal degenerative diseases of the central or peripheral nervoussystem, by administering to a mammal suffering from such a disease anamount of a prosaposin fragment effective to retard or halt neuronaldegeneration. Most preferably, this fragment includes the neurotrophicactivity of the peptide of SEQ ID NO 1 and is able to cross the bloodbrain barrier. In addition, the administration is intravenous orintradermal. It is envisioned that the disease is a central nervoussystem disorder including Alzheimer's disease, Parkinson's disease,stroke, post-polio syndrome and amyotrophic lateral sclerosis.

Further, the invention includes a method for retarding the progress ofretinal neuropathy (senile macular degeneration) in a patient byadministering to the patient an effective amount of prosaposin or aneurotrophic fragment thereof. Preferably this retinal neuropathy issenile macular degeneration, the patient is a human over the age of 65,and the administration is either topical, intravenous or intraocular.

Another aspect of the present invention is a pharmaceutical compositioncomprising prosaposin or a neurotrophic fragment thereof in unit dosageform.

Still another aspect of the present invention is a pharmaceuticalcomposition comprising prosaposin or a neurotrophic fragment thereofformulated with a controlled release material.

Finally, the invention includes a neural prosaposin receptor protein inisolated or purified form. Preferably, this receptor protein is the sameprotein that can be isolated from a P100 plasma membrane fraction byaffinity purification using a neurite growth-inducing peptide containedwithin the saposin C sequence linked to a solid support, and has amolecular weight of approximately 20 kDa.

DETAILED DESCRIPTION OF THE INVENTION

This invention discloses our discovery that prosaposin, saposin C or apeptide comprising amino acids 8-29 of saposin C can be used tostimulate neurite outgrowth and to promote increased myelination.

Prosaposin or its derivatives possess significant therapeuticapplications in promoting functional recovery after toxic, traumatic,ischemic, degenerative and inherited lesions to the peripheral andcentral nervous system. In addition, prosaposin or its derivatives maybe used to counteract the effects of demyelinating diseases.

Prosaposin and its derivatives are known to be present in many types ofneurons, are water soluble (in contrast to glycosphingolipids) and areless immunogenic than ganglioside micelles since for therapy in humansthe human sequence will be used which will not elicit an immuneresponse.

Human prosaposin has the amino acid sequence set forth in SEQ ID NO: 2.Saposin C has the amino acid sequence set forth in SEQ ID NO: 3. Thehuman cDNA sequence for prosaposin is set forth in SEQ ID NO: 4. Anactive 18-mer fragment derived from the active 22-mer fragment (SEQ IDNO: 1) is set forth as SEQ ID NO: 5.

As will be discussed in more specific detail in the Examples,prosaposin, saposin C and amino acids of saposin C that include at leastamino acids 8-29 (SEQ ID NO: 3) are active as neurotrophic factors. Inaddition, a peptide including at least amino acids 12-29 (with atyrosine substituted for valine at position 12) SEQ ID NO: 5 is also anactive neurotrophic factor. Similar active peptides, also within thescope of this invention, can be prepared and screened as describedherein (See Example 2). These proteins and peptides stimulate theoutgrowth of neurites, promote myelination and prevent programmed celldeath in neuronal tissues.

One aspect of the present invention is a method for facilitatingoutgrowth of neurites in differentiated or undifferentiated neuralcells. This method requires administration of an effective,neurite-outgrowth facilitating amount of prosaposin, saposin C, or the18 or 22 amino acid fragment thereof to the cells in question. A typicalminimum amount of prosaposin for the neurotrophic factor activity incell growth medium is usually at least about 1.4×10⁻¹¹ M, or about 10ng/ml. This amount or more of saposin C or its active 18 or 22 aminoacid fragments may also be used. Usually concentrations in the range of0.1 μg/ml to about 10 μg/ml of any of these materials will be used.Effective amounts for any particular tissue can be determined inaccordance with Example 1.

The neural cells can be treated in vitro or ex vivo by directlyadministering the neurotrophic factor of the present invention to thecells. This can be done, for example, by culturing the cells in growthmedium suitable for the particular cell type followed by addition of theneurotrophic factor to the medium.

When the cells to be treated are in vivo, typically in a vertebrate,preferably a mammal or a bird, the composition can be administered tothe cells to be treated by one of several techniques. Most preferably,the composition can be injected directly into the blood in sufficientquantity to give the desired concentration of neurotrophic factor sincean iodinated 18-mer peptide consisting of amino acids 12-29 of the22-mer with a substitution of tyrosine for valine at amino acid 12(M.W=2000) crosses the blood brain barrier and enters the centralnervous system as described in Example 7 (see Banks et al., (1992)Peptides, 13: 1289-1294). The uptake by the brain was approximately0.03% which is in the midrange of values for peptides of thatapproximate size which will cross the blood brain barrier. This is theonly neurotrophic factor so far described which will cross the bloodbrain barrier when administered intravenously.

Direct intracranial injection or injection into the cerebrospinal fluidmay also be used in sufficient quantities to give the desired localconcentration of neurotrophin. In both cases, a pharmaceuticallyacceptable injectable carrier of well known type can be used. Suchcarriers include, for example, phosphate buffered saline (PBS).Alternatively, the composition can be administered to peripheral neuraltissue by direct local injection or by systemic administration. Variousconventional modes of administration are contemplated, includingintravenous, intramuscular, intradermal, subcutaneous, intracranial,epidural, topical and oral administration.

The composition can be packaged and administered in unit dosage formsuch as an injectable composition or local preparation in a dosageamount equivalent to the daily dosage administered to a patient or as acontrolled release composition. A septum sealed vial containing a dailydose of the active ingredient in either PBS or in lyophilized form is anexample of a unit dosage.

Since the molecular weight of the active 22-mer is approximately 2600,and an iodinated 18-mer contained within this sequence will cross theblood brain barrier, then the 22-mer will also most likely cross andenter the central nervous system (Banks et al., (1992) Peptides, 13:1289-1294). Appropriate daily systemic dosages based on the body weightof the vertebrate are in the range of from about 10 to about 100 μg/kg,although dosages from about 0.1 to about 1000 μg/kg are alsocontemplated. Daily dosages of locally administered material will beabout an order of magnitude less. Oral administration may be possible ifthe peptide is stable to gastrointestinal degradation and readilyabsorbed.

In one preferred embodiment of the invention, the neurotrophic factor isadministered locally to the neural cells in vivo by implantation of thematerial. For example, polylactic acid, polygalactic acid, regeneratedcollagen, multilamellar liposomes and many other conventional depotformulations comprise bioerodible or biodegradable materials that can beformulated with biologically active compositions. These materials, whenimplanted, gradually break down and release the active material to thesurrounding tissue. The use of bioerodible, biodegradable and otherdepot formulations is expressly contemplated in the present invention.Infusion pumps, matrix entrapment systems, and combination withtransdermal delivery devices are also contemplated.

The neurotrophic factors of the present invention may alsoadvantageously be enclosed in micelles or liposomes. Liposomeencapsulation technology is well known. Liposomes may be targeted tospecific tissue, such as neural tissue, through the use of receptors,ligands or antibodies capable of binding the targeted tissue. Thepreparation of these formulations is well known in the art (i.e., Radinand Metz, (1983) Methods Enzymol. 98: 613-618).

There are currently no available pharmaceuticals able to promote fullfunctional regeneration and restoration of structural integrity ofneural systems. This is particularly true of the central nervous system.Regeneration of peripheral nerves through use of neurotrophic factors isa more immediately demonstrable goal. Such treatment is within the scopeof this invention. Moreover, neurotrophic factors can be therapeuticallyuseful in the treatment of neurodegenerative diseases associated withthe degeneration of neural populations or specific areas of the brain.The principal cause of Parkinson's disease is the degeneration ofdopaminergic neurons of the substantia nigra. Since antibodies againstprosaposin immunohistochemically stain the dopaminergic neurons of thesubstantia nigra in human brain sections, prosaposin and its activefragments may be therapeutically useful in the treatment of Parkinson'sdisease.

It has long been believed that in order to reach neuronal populations inthe brain, neurotrophic factors would have to be administeredintracerebrally, since these proteins do not cross the blood-brainbarrier. However, as previously mentioned, the active iodinated 18-merwill cross and the active 22-mer will most likely cross this barrier andwould thus be administered intravenously. Other neuronal populations,such as motor neurons, would also be treated by intravenous injection,although direct injection into the cerebrospinal fluid is alsoenvisioned as an alternate route.

Cells may be treated to facilitate myelin formation or to preventdemyelination in the manner described above, both in vitro, ex vivo andin vivo. There are several diseases that result in demyelination ofnerve fibers including multiple sclerosis, acute disseminatedleukoencephalitis, progressive multifocal leukoencephalitis,metachromatic leukodystrophy and adrenal leukodystrophy. These diseasescan be treated, and the progression of the demyelination can be slowedor halted, by administration of the neurotrophic factors of the presentinvention to the cells affected by the disease.

The compositions of the present invention can be used in vitro asresearch tools for studying the effects of neurotrophic factors andmyelin facilitating materials. However, more practically, they have animmediate use as laboratory reagents and components of cell growth mediain order to better enable growth of neural cells in vitro.

The prosaposin used in the present invention may be obtained fromvarious sources, and may be, for example, naturally occurring proteinisolated from human milk or seminal plasma or recombinant humanprosaposin purified from spent media of Spodoptera frugiperda (Sf9)cells infected with a baculovirus expression vector containingfull-length cDNA for human prosaposin as described (Dewji et al., (1987)Proc. Natl. Acad. Sci. USA, 84: 8652-8656). O'Brien et al., (1988)Science, 241: 1098-1101); Hitaiwa et al., (1993) Arch. Biochem.Biophys., 304, 110-116). Saposin C is isolated in pure form from spleensof patients with Gaucher disease, a lysosomal storage disorder (Morimotoet al., (1990) Proc. Natl. Acad. Sci. USA, 87: 3493-3497). Saposin C (80amino acids) can also be chemically synthesized and refolded (Weiler etal., (1993) J. Mol. Neurosci., in press).

The peptides corresponding to sequences within saposin C may besynthesized using an automated solid-phase protocol on an AppliedBiosystems Model 430 peptide synthesizer. After synthesis, peptides1-40, 41-82, 1-27, 13-34 and 21-48 are desalted on a Sephadex G-75column prior to use.

EXAMPLE 1 Effect of prosaposin, saposins and NGF on NS20Y neuriteoutgrowth

NS20Y neuroblastoma cells were grown in Dulbecco's Modified Eagle Medium(DMEM) containing 10% fetal calf serum (FCS) and 1 mM sodium pyruvate.Cells were removed with trypsin and plated in 30 mm petra dishes ontoglass coverslips. After 20-24 hours the medium was replaced with DMEMcontaining 0.5% fetal calf serum plus effector proteins. Cells werecultured for another 24 hours, washed with phosphate buffered saline(PBS) and fixed with Bouin's solution (saturated aqueous picricacid/formalin/acetic acid 15:5:1) for 30 minutes. Fixative was removedwith PBS and neurite outgrowth was scored under a phase contrastmicroscope. Cells exhibiting one or more clearly defined neurites equalto or longer than one cell diameter were scored as positive. At least200 cells were scored in different portions of each dish to determinethe percentage of neurite bearing cells and assays were performed induplicate.

A dose-response curve (FIG. 1a) demonstrated that prosaposin promotedreversible neurite outgrowth in NS20Y neuroblastoma cells. The lowestconcentration for activity was 1.4×10⁻¹¹ M (10 ng/ml) which is in theeffective concentration range of other neurotrophins. When prosaposinwas removed, retraction of neurite outgrowth was complete at 36 hours,demonstrating that its continual presence is necessary in order tomaintain neurite outgrowth. In addition, saposin C was the sole fragmentof prosaposin found to possess neuritogenic activity, as did the 22-merand iodinated 18-mer peptides derived from the saposin C sequence.

Since nerve growth factor (NGF) acts on a variety of cell types, wewanted to determine whether it was involved in prosaposin-mediatedoutgrowth in neuroblastoma cells. NGF by itself had no effect on neuriteoutgrowth in NS20Y cells and did not augment the prosaposin response(FIG. 1b). When 5'-methyladenosine (MeSAdo), which specifically inhibitsNGF-induced neuritogenesis in PC12M pheochromocytoma cells was added,MeSAdo did not inhibit prosaposin-induced NS20Y neurite outgrowth.Additionally, prosaposin failed to stimulate neurite outgrowth fromNGF-responsive PC12M cells at high concentrations (2 mg/ml). Since NS20Ycells are not NGF responsive, this indicates that the NGF response andthe prosaposin response are different.

A set of synthetic peptides from different regions of saposin C wasutilized to further define the active sequence. An amino terminalpeptide (1-40) was active and a carboxy terminal peptide (41-82) wasinactive. Testing of four more peptides (Table 2) further narrowed theactive sequence to a region between residues 8-29, the most hydrophilicregion in the saposin C domain (FIG. 2a) which also contains the singleglycosylation site (Asn 22). Higher concentrations of the active 22-mer(residues 8-29) were required for activity but the extent of neuriteoutgrowth was greater than with prosaposin or saposin C (FIG. 1a). Thesequence between residues 18 and 29 is highly conserved (FIG. 2b).Interestingly, human samosin A is nearly identical to saposin C in thisregion except for the first four residues, indicating that the activesequence requires the presence of leucine 18 and asparagines at residues21 and 22 or both.

                  TABLE 2                                                         ______________________________________                                        Neurite outgrowth response of NS20Y cells treated with                        human saposin C, saposin A and synthetic peptides from the                    human saposin C domain at 5 μg/ml. The dose response curve                 for peptide 8-29 (active 22 mer) is given in FIG. 1a.                         Peptide Added (5 μg/ml)                                                                    % Neurites after 24 hours                                     ______________________________________                                        Saposin C       40%                                                           1-40            42%                                                           41-82           17%                                                           1-27            46%                                                           13-34           50%                                                           21-48           18%                                                           8-29            56%                                                           Saposin A       20%                                                           None            18%                                                           ______________________________________                                    

To test whether gangliosides were involved in the response, aprosaposin-ganglioside GM1 complex (4:1) was generated by a method wellknown in the art. When tested in the neurite outgrowth assay, thecomplex had negligible activity. The same result was obtained with aganglioside GM3-saposin C complex. This indicated that the neuritogeniceffect was not the result of ganglioside transport, but was instead dueto the prosaposin and saposin C, respectively.

In order to determine whether prosaposin or its fragments would have aneffect on neurite outgrowth in nontransformed cells, newborn mousecerebellar explants were used as described in the following example:

EXAMPLE 2 Effect of prosaposin and its active fragments on neuriteoutgrowth in mouse cerebellar explants

Newborn mouse cerebellar explants were prepared according to Satomi(Zool. Sci. 9, 127-137 (1992)). Neurite outgrowth and myelination wereobserved over 22 days in culture, during the period when the newbornmouse cerebellum normally undergoes neuronal differentiation andmyelination begins. Prosaposin (5 μg/ml) and saposins A, B and C (10μg/ml) were added on the second day after preparation of the explants(three control and three treated explants) and outgrowth of neurites andmyelination were assessed under a bright field microscope with a videocamera. On the eighth day cultures containing prosaposin and saposin Cbecame thinner and more spread out than control cultures. On day 15, theprosaposin and saposin C treated cultures contained many cells with longprojections at the periphery of the explant which were less prominent incontrols or those treated with saposins A or B. Saposin C treatedcultures contained twice as many myelinated axons in the subcorticalwhite matter at 22 days as controls or those treated with saposins A orB. Both the number of myelinated fibers observed visually per opticalfield and the activity of the myelin marker enzyme CNP were twice thecontrol value. These results demonstrate that the neurotrophic effect ofprosaposin and saposin C also occurs in differentiating cerebellum exvivo. These results further demonstrate the ability of prosaposin andsaposin C to induce increased myelination in differentiating cerebellumex vivo.

Since prosaposin appears to be active at the plasma membrane it shouldbe present in the plasma membranes of responsive cells as shown in thefollowing example:

EXAMPLE 3 Western blots of prosaposin and saposin C from NS20Y cells

NS20Y cells were grown to confluence in 75 cm flasks in the presence ofgrowth medium. Cells were harvested by scraping and surface membraneswere isolated by the zinc ion method of Warren and Glick (1969) usingdiscontinuous gradients of 50, 48, 45, 43, 40 and 35% sucrose; surfacemembranes localize in the 40 and 43% sucrose fraction. These fractions,as well as the infranatant and supernatant fractions bounding them, wereelectrophoresed on 10% SDS polyacrylamide gels along with the whole cellextracts, transferred to nitrocellulose filters, and probed with amonoclonal antibody to saposin C by methods well known in the art.

Examination of Western blots revealed that prosaposin, migrating as a 68kDa band on SDS polyacrylamide gels, was localized to surface membranefractions from both NS20Y and Neuro 2A cells. Mature saposin C andintermediate molecular weight saposin derivatives were minor componentsof the membrane fractions but were abundant in the whole cell extract.This demonstrates that prosaposin is located in the plasma membrane ofresponsive cells.

In order to localize prosaposin histochemically, neuroblastoma celllines were immunostained with a prosaposin-specific antibody (JP-1) asillustrated in the following example:

EXAMPLE 4 Immunohistochemical localization of propsaposin

Cells were grown on glass cover slips, washed three times with PBS andfixed with Bouin's solution for one hour at room temperature. Bouin'ssolution was then rinsed out with 5 washes of PBS and slips wereincubated in 30% goat serum, 0.5% Tween 20 in PBS to block nonspecificbinding and, after rinsing, were incubated in a 1:100 dilution of IgGpurified rabbit JP-1 at 4° C. overnight. After rinsing with PBScontaining 0.1% Triton X-100, the preparations were incubated witheither peroxidase conjugated goat anti-rabbit IgG (Bio-Rad, 1:2000) orFITC-conjugated goat anti-rabbit IgG (Cappel, 1:2000). After rinsing,peroxidase immunostaining was detected using theimidazole-diaminobenzidine-H₂ O₂ reaction. Fluorescence immunostainingwas detected under a fluorescence microscope using Nofade as a quenchingdeterrent. Preimmune rabbit IgG (1:100) was used as a control fornonspecific binding. Immunostaining of extended neurites, plasmamembranes and growth cones were observed.

A similar methodology was used to immunostain postmortem human brainsections to detect reactive cell types. In frontal cortex, intensestaining of the perikarya of large and medium sized Golgi type 1 neuronswas observed. The surface of neuronal perikarya and the proximal segmentof axons at the hillock region were also strongly stained as were someextended axons. In the cerebellum strong staining of Purkinje andstellate cells was observed, as well as large neurons in the cerebellarnuclei (dentate, emboliform and globose nuclei). Cerebellar granularcells were moderately stained. In the mesencephalon, moderate stainingwas observed in dopaminergic neurons of the substantia nigra. Largeneurons in the red nucleus, neurons in the oculomotor nucleus, theamygdaloid nucleus and ependymal cells lining the lateral ventricle werealso moderately stained. In the hippocampus, pyramidal cells and granulecells of the dentate gyrus were strongly stained. In the spinal cordalpha motor neurons were intensely stained. This survey indicated thatprosaposin was localized to populations of large neurons including upperand lower motor neurons.

Since all neurotrophins identified thus far exert their effects bybinding to a cell surface receptor and initiating a kinase cascade,phosphorylation assays were performed in NS20Y cells treated withprosaposin or its fragments as described in the following example:

EXAMPLE 5 Incorporation of 32P into NS20Y proteins after treatment withprosaposin or its active fragments

NS20Y cells were incubated in phosphate-free Hanks' balanced saltsolution containing 2.5 μg/ml actinomycin D and 80-100 μCi/mlcarrier-free ³² P!-orthophosphate (New England Nuclear) and effectorproteins (0.5-1.0 μg/ml) and incubated for 10-15 minutes at roomtemperature. Cells were solubilized in SDS-PAGE sample buffer, analyzedby SDS-PAGE and autoradiographed.

Prosaposin, saposin C and SEQ ID NO: 1 were found to stimulatephosphorylation of proteins of 148, 100, 80, 68, 50, 38 and 34 kDa to agreater extent than controls or cells treated with similarconcentrations of saposins A, B or D. This 148 kDa protein may bephospholipase C-γ, a protein known to be involved in phospholipidmetabolism and which is phosphorylated on tyrosine residues in responseto a number of growth factors. Densitometric analysis indicated a 3-5fold stimulation of phosphorylation after 10 minutes. Treatment of gelswith alkali revealed that the prominent phosphorylated proteins werealkali-resistant, indicating that they contain phosphotyrosine and/orphosphothreonine (located next to proline) residues. These resultsindicate that prosaposin and its active fragments bind to a cell surfacereceptor and activate a kinase cascade, similar to other neurotrophinsand growth factors.

Since prosaposin-ganglioside GM1 or saposin C-ganglioside GM3 complexesinhibit neuritogenesis, while prosaposin or saposin C alone promote thisprocess, this indicates that gangliosides may abolish neurotogenicactivity by masking a receptor binding site on the neurotrophin. Inaddition, since prosaposin and its active fragments induce tyrosinephosphorylation of cytoplasmic proteins in responsive cells, most likelyby activation of a tyrosine kinase(s) similar to cytokines and growthfactors, this provides further evidence that a cell surface receptor isinvolved.

A 20 kDa protein has been identified as the putative receptor forprosaposin as described in the following example:

EXAMPLE 6 Isolation of the prosaposin receptor

The putative prosaposin receptor protein was isolated from whole ratbrain, rat cerebellum and mouse neuroblastoma cells using the plasmamembrane P-100 fraction. Briefly, cells or tissues were solubilized andcentrifuged at 14,000 rpm to remove debris. The supernatant wascentrifuged at 40,000 rpm for 1 hour at 4° C. The pellet, enriched inplasma membrane, was solubilized in RIPA buffer (10 mM MOPS, pH 7.5,0.3M sucrose, 5 mM EDTA, 1% Trasylol, 10 μM leupeptin and 10 μMantipain). This P-100 fraction was applied to an affinity columncontaining the bound, active 22-mer fragment of saposin C. The columnwas washed with 0.25M NaCl to elute loosely-bound proteins followed by1.0M NaCl which eluted the putative 20 kDa prosaposin receptor. Inaddition, it was determined that the 20 kDa protein could be elutedusing a 100 fold excess of unbound peptide thus demonstrating specificelution. The 20 kDa protein was approximately 90% pure as judged bySDS-PAGE. The protein was purified to homogeneity using HPLC and elutedat 50% acetonitrile in an acetonitrile/water gradient on a Vydac C4column.

EXAMPLE 7 In vivo peptide uptake by the central nervous system

An 18-mer peptide consisting of amino acids 12-29 of saposin C with atyrosine substituted for valine at position 12 was chemicallysynthesized on an Applied Biosystems Model 430 peptide synthesizer. Thepeptide was then radioiodinated by the lactoperoxidase method and 20×10⁶cpm were injected into the auricles of rats. The animals were sacrificedafter one hour and 24 hours and the hearts were perfused with isotonicsaline in order to remove the blood from the brain. The brain was thencounted in a gamma counter in order to determine the percentage ofpeptide uptake. In addition, in the 24 hour experiment the brain washomogenized and separated into a capillary rich fraction (pellet) and aparenchymal brain fraction (supernatant) after dextran centrifugation(Triguero et al., (1990) J. Neurochem., 54: 1882-1888). This methodallows for the discrimination between radiolabelled peptide within bloodvessels and that within the brain. In the 24 hour experiment, 0.017% ofthe injected peptide was detected in whole brain; 75% of the label wasin the parenchymal fraction and 25% was in the capillary fraction. At 1hour 0.03% of the injected dose was present in whole brain.

EXAMPLE 8 Use of prosaposin and its active fragments in treatingtraumatic ischemic lesions to the CNS in vivo

Rats with traumatic lesions to the spinal cord receive direct orintravenous administration of prosaposin or its active fragments in the10 ng-10 mg/ml range in a sterile saline solution or in a depot form toenable slow release. The same number of animals receive only saline.After surgical partial transection of the spinal cord or a crush injury,prosaposin or a neurotrophic fragment thereof is directly injected intothe lesion site using the same dose range (control animals receivesaline injections) and improvement is assessed by gain of motor neuronfunction (i.e., increased limb movement). The treatments continue untilno further improvement occurs. Since prosaposin and its active fragmentsare very water-soluble, no special delivery system for the preparationis required. Injection of the 18 or 22 amino acid fragments is preferredsince there is less chance of degradation and diffusion will be greater.Additionally, this fragment can be chemically synthesized in largequantities.

EXAMPLE 9 Use of prosaposin and its active fragments in treatingdemyelination disorders

Patients diagnosed with early stage MS (or other demyelination disorder)are given the active 18 or 22-mer fragment (in saline) by directintravenous injection or injection into the cerebrospinal fluid usingthe same dose range as in Example 7. Control patients receive onlysaline. The treatment is administered weekly or monthly and anyimprovement is assessed by increased muscle strength, musculoskeletalcoordination, and assessing myelination by magnetic resonance imaging.

EXAMPLE 10 Use of prosaposin or its active fragments in treating retinalneuropathy

Retinal neuropathy, an ocular neurodegenerative disorder leading to lossof vision in the elderly, is believed to be a disorder treatable byprosaposin or its active fragments. Prosaposin or its activeneurotrophic fragments are administered either topically, systemicallyor intraocularly in an amount sufficient to produce a localconcentration of neurotrophin of about 10 ng/ml to about 10 mg/ml. Theadministration is continued weekly until visual loss is slowed or nofurther increase in vision is noticed.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 11                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CysGluPheLeuValLysGluValThrLysLeuIleAspAsnAsnLys                              151015                                                                        ThrGluLysGluIleLeu                                                            20                                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 523 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetTyrAlaLeuPheLeuLeuAlaSerLeuLeuGlyAlaAlaLeuAla                              151015                                                                        GlyProValLeuGlyLeuLysGluCysThrArgGlySerAlaValTrp                              202530                                                                        CysGlnAsnValLysThrAlaSerAspCysGlyAlaValLysHisCys                              354045                                                                        LeuGlnThrValTrpAsnLysProThrValLysSerLeuProCysAsp                              505560                                                                        IleCysLysAspValValThrAlaAlaGlyAspMetLeuLysAspAsn                              65707580                                                                      AlaThrGluGluGluIleLeuValTyrLeuGluLysThrCysAspTrp                              859095                                                                        LeuProLysProAsnMetSerAlaSerCysLysGluIleValAspSer                              100105110                                                                     TyrLeuProValIleLeuAspIleIleLysGlyGluMetSerArgPro                              115120125                                                                     GlyGluValCysSerAlaLeuAsnLeuCysGluSerLeuGlnLysHis                              130135140                                                                     LeuAlaGluLeuAsnHisGlnLysGlnLeuGluSerAsnLysIlePro                              145150155160                                                                  GluLeuAspMetThrGluValValAlaProPheMetAlaAsnIlePro                              165170175                                                                     LeuLeuLeuTyrProGlnAspGlyProArgSerLysProGlnProLys                              180185190                                                                     AspGlyAspValCysGlnAspCysIleGlnMetValThrAspIleGln                              195200205                                                                     ThrAlaValArgThrAsnSerThrPheValGlnAlaLeuValGluHis                              210215220                                                                     ValLysGluGluCysAspArgLeuGlyProGlyMetAlaAspIleCys                              225230235240                                                                  LysAsnTyrIleSerGlnTyrSerGluIleAlaIleGlnMetMetMet                              245250255                                                                     HisMetGlnProLysGluIleCysAlaLeuValGlyPheCysAspGlu                              260265270                                                                     ValLysGluMetProMetGlnThrLeuValProAlaLysValAlaSer                              275280285                                                                     LysAsnValIleProAlaLeuAspLeuValAspProIleLysLysHis                              290295300                                                                     GluValProAlaLysSerAspValTyrCysGluValCysGluPheLeu                              305310315320                                                                  ValLysGluValThrLysLeuIleAspAsnAsnLysThrGluLysGlu                              325330335                                                                     IleLeuAspAlaPheAspLysMetCysSerLysLeuProLysSerLeu                              340345350                                                                     SerGluGluCysGlnGluValValAspThrTyrGlySerSerIleLeu                              355360365                                                                     SerIleLeuLeuGluGluValSerProGluLeuValCysSerMetLeu                              370375380                                                                     HisLeuCysSerGlyThrArgLeuProAlaLeuThrValHisValThr                              385390395400                                                                  GlnProLysAspGlyGlyPheCysGluValCysLysLysLeuValGly                              405410415                                                                     ThrLeuAspArgAsnLeuGluLysAsnSerThrLysGlnGluIleLeu                              420425430                                                                     AlaAlaLeuGluLysGlyCysSerPheLeuProAspProTyrGlnLys                              435440445                                                                     GlnCysAspGlnPheValAlaGluTyrGluProValLeuIleGluIle                              450455460                                                                     LeuValGluValMetAspProSerPheValCysLeuLysIleGlyAla                              465470475480                                                                  CysProSerAlaHisLysProLeuLeuGlyThrGluLysCysIleTrp                              485490495                                                                     GlyProSerTyrTrpCysGlnAsnThrGluThrAlaAlaGlnCysAsn                              500505510                                                                     AlaValGluHisCysLysArgHisValTrpAsn                                             515520                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 80 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       SerAspValTyrCysGluValCysGluPheLeuValLysGluValThr                              151015                                                                        LysLeuIleAspAsnAsnLysThrGluLysGluIleLeuAspAlaPhe                              202530                                                                        AspLysMetCysSerLysLeuProLysSerLeuSerGluGluCysGln                              354045                                                                        GluValValAspThrTyrGlySerSerIleLeuSerIleLeuLeuGlu                              505560                                                                        GluValSerProGluLeuValCysSerMetLeuHisLeuCysSerGly                              65707580                                                                      (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2740 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       ATGTACGCCCTCTTCCTCCTGGCCAGCCTCCTGGGCGCGGCTCTAGCCGGCCCGGTCCTT60                GGACTGAAAGAATGCACCAGGGGCTCGGCAGTGTGGTGCCAGAATGTGAAGACGGCGTCC120               GACTGCGGGGCAGTGAAGCACTGCCTGCAGACCGTTTGGAACAAGCCAACAGTGAAATCC180               CTTCCCTGCGACATATGCAAAGACGTTGTCACCGCAGCTGGTGATATGCTGAAGGACAAT240               GCCACTGAGGAGGAGATCCTTGTTTACTTGGAGAAGACCTGTGACTGGCTTCCGAAACCG300               AACATGTCTGCTTCATGCAAGGAGATAGTGGACTCCTACCTCCCTGTCATCCTGGACATC360               ATTAAAGGAGAAATGAGCCGTCCTGGGGAGGTGTGCTCTGCTCTCAACCTCTGCGAGTCT420               CTCCAGAAGCACCTAGCAGAGCTGAATCACCAGAAGCAGCTGGAGTCCAATAAGATCCCA480               GAGCTGGACATGACTGAGGTGGTGGCCCCCTTCATGGCCAACATCCCTCTCCTCCTCTAC540               CCTCAGGACGGCCCCCGCAGCAAGCCCCAGCCAAAGGATAATGGGGACGTTTGCCAGGAC600               TGCATTCAGATGGTGACTGACATCCAGACTGCTGTACGGACCAACTCCACCTTTGTCCAG660               GCCTTGGTGGAACATGTCAAGGAGGAGTGTGACCGCCTGGGCCCTGGCATGGCCGACATA720               TGCAAGAACTATATCAGCCAGTATTCTGAAATTGCTATCCAGATGATGATGCACATGCAA780               CCCAAGGAGATCTGTGCGCTGGTTGGGTTCTGTGATGAGGTGAAAGAGATGCCCATGCAG840               ACTCTGGTCCCCGCCAAAGTGGCCTCCAAGAATGTCATCCCTGCCCTGGAACTGGTGGAG900               CCCATTAAGAAGCACGAGGTCCCAGCAAAGTCTGATGTTTACTGTGAGGTGTGTGAATTC960               CTGGTGAAGGAGGTGACCAAGCTGATTGACAACAACAAGACTGAGAAAGAAATACTCGAC1020              GCTTTTGACAAAATGTGCTCGAAGCTGCCGAAGTCCCTGTCGGAAGAGTGCCAGGAGGTG1080              GTGGACACGTACGGCAGCTCCATCCTGTCCATCCTGCTGGAGGAGGTCAGCCCTGAGCTG1140              GTGTGCAGCATGCTGCACCTCTGCTCTGGCACGCGGCTGCCTGCACTGACCGTTCACGTG1200              ACTCAGCCAAAGGACGGTGGCTTCTGCGAAGTGTGCAAGAAGCTGGTGGGTTATTTGGAT1260              CGCAACCTGGAGAAAAACAGCACCAAGCAGGAGATCCTGGCTGCTCTTGAGAAAGGCTGC1320              AGCTTCCTGCCAGACCCTTACCAGAAGCAGTGTGATCAGTTTGTGGCAGAGTACGAGCCC1380              GTGCTGATCGAGATCCTGGTGGAGGTGATGGATCCTTCCTTCGTGTGCTTGAAAATTGGA1440              GCCTGCCCCTCGGCCCATAAGCCCTTGTTGGGAACTGAGAAGTGTATATGGGGCCCAAGC1500              TACTGGTGCCAGAACACAGAGACAGCAGCCCAGTGCAATGCTGTCGAGCATTGCAAACGC1560              CATGTGTGGAACTAGGAGGAGGAATATTCCATCTTGGCAGAAACCACAGCATTGGTTTTT1620              TTCTACTTGTGTGTCTGGGGGAATGAACGCACAGATCTGTTTGACTTTGTTATAAAAATA1680              GGGCTCCCCCACCTCCCCCATTTCTGTGTCCTTTATTGTAGCATTGCTGTCTGCAAGGGA1740              GCCCCTAGCCCCTGGCAGACATAGCTGCTTCAGTGCCCCTTTTCTCTCTGCTAGATGGAT1800              GTTGATGCACTGGAGGTCTTTTAGCCTGCCCTTGCATGGCGCCTGCTGGAGGAGGAGAGA1860              GCTCTGCTGGCATGAGCCACAGTTTCTTGACTGGAGGCCATCAACCCTCTTGGTTGAGGC1920              CTTGTTCTGAGCCCTGACATGTGCTTGGGCACTGGTGGGCCTGGGCTTCTGAGGTGGCCT1980              CCTGCCCTGATCAGGGACCCTCCCCGCTTTCCTGGGCCTCTCAGTTGAACAAAGCAGCAA2040              AACAAAGGCAGTTTTATATGAAAGATTAGAAGCCTGGAATAATCAGGCTTTTTAAATGAT2100              GTAATTCCCACTGTAATAGCATAGGGATTTTGGAAGCAGCTGCTGGTGGCTTGGGACATC2160              AGTGGGGCCAAGGGTTCTCTGTCCCTGGTTCAACTGTGATTTGGCTTTCCCGTGTCTTTC2220              CTGGTGATGCCTTGTTTGGGGTTCTGTGGGTTTGGGTGGGAAGAGGGCAATCTGCCTGAA2280              TGTAACCTGCTAGCTCTCCGAAGGCCCTGCGGGCCTGGCTTGTGTGAGCGTGTGGACAGT2340              GGTGGCCGCGCTGTGCCTGCTCGTGTTGCCTACATGTCCCTGGCTGTTGAGGCGCTGCTT2400              CAGCCTGCACCCCTCCCTTGTCTCATAGATGCTCCTTTTGACCTTTTCAAATAAATATGG2460              ATGGCGAGCTCCTAGGCCTCTGGCTTCCTGGTAGAGGGCGGCATGCCGAAGGGTCTGCTG2520              GGTGTGGATTGGATGCTGGGGTGTGGGGGTTGGAAGCTGTCTGTGGCCCACTTGGGCACC2580              CACGCTTCTGTCCACTTCTGGTTGCCAGGAGACAGCAAGCAAAGCCAGCAGGACATGAAG2640              TTGCTATTAAATGGACTTCGTGATTTTTGTTTTGCACTAAAGTTTCTGTGATTTAACAAT2700              AAAATTCTGTTAGCCAGAAAAAAAAAAAAAAAAAAAAAAA2740                                  (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       TyrLysGluValThrLysLeuIleAspAsnAsnLysThrGluLysGlu                              151015                                                                        IleLeu                                                                        (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       CysGlnPheValMetAsnLysPheSerGluLeuIleValAsnAsnAla                              151015                                                                        ThrGluGluLeuLeuTyr                                                            20                                                                            (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       CysGlnLeuValAsnArgLysLeuSerGluLeuIleIleAsnAsnAla                              151015                                                                        ThrGluGluLeuLeu                                                               20                                                                            (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       CysGluTyrValValLysLysValMetLeuLeuIleAspAsnAsnArg                              151015                                                                        ThrGluGluLysIleIle                                                            20                                                                            (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       CysGluPheValValLysGluValAlaLysLeuIleAspAsnAsnArg                              151015                                                                        ThrGluGluGluIleLeu                                                            20                                                                            (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Other                                                           (B) LOCATION: 21...21                                                         (D) OTHER INFORMATION: Xaa=I or L                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      CysXaaXaaXaaValXaaXaaXaaXaaXaaLeuIleXaaAsnAsnXaa                              151015                                                                        ThrGluXaaXaaXaaXaa                                                            20                                                                            (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      CysLysAspValValThrAlaAlaGlyAspMetLeuLysAspAsnAla                              151015                                                                        ThrGluGluGluIleLeu                                                            20                                                                            __________________________________________________________________________

What is claimed is:
 1. A method for treating demyelination in a mammalcomprising:administering to a mammal afflicted with demyelination apharmaceutically effective demyelination inhibiting mount of prosaposinor a neurotrophic fragment comprising amino acids 8-29 of SEQ ID NO: 3,or including the active neurotrophic fragment located within amino acids8-29 of SEQ ID NO:
 3. 2. The method of claim 1 wherein said fragmentcomprises saposin C.
 3. The method of claim 1, wherein saiddemyelination is due to multiple sclerosis, ischemic injury or traumaticinjury.
 4. The method of claim 1 wherein said administration is selectedfrom the group consisting of: intravenous, intramuscular, intradermal,subcutaneous, intracranial, intracerebrospinal and topical.
 5. Themethod of claim 1 wherein said prosaposin or fragment thereof isadministered in a biologically compatible carrier.
 6. The method ofclaim 1 wherein said prosaposin or fragment thereof is enclosed in alamellar structure.
 7. A method for inhibiting the progress of neural ormyelin degeneration in neural tissue, comprising:contacting neuraltissue susceptible to such degeneration with prosaposin or aneurotrophic fragment comprising amino acids 8-29 of SEQ ID NO: 3, orincluding the active neurotrophic fragment located within amino acids8-29 of SEQ ID NO:
 3. 8. The method of claim 7 wherein said fragment issaposin C.
 9. The method of claim 7 wherein said tissue is in vitro. 10.The method of claim 7 wherein said tissue is in vivo.